Project Summary/Abstract The gastrointestinal (GI) tract is populated by a dense and diverse microbiota that impacts human health. Although the microbiota composition at the species level for each individual is unique as a fingerprint, its composition at the phyla level is more conserved. The predominant phyla are Bacteroidetes and Firmicutes, followed by Proteobacteria. The gut microbiota has been largely regarded as a resistance barrier towards enteric pathogens. However, the enteric pathogens that cause infectious colitis, enterohemorrhagic E. coli (EHEC) O157:H7 and Citrobacter rodentium (extensively used as a surrogate EHEC model for murine infections, given that EHEC does not infect mice), exploit cues and nutrients made available by members of the microbiota to regulate their virulence program. They sense several metabolites, including sugar sources such as fucose, and organic acids such as succinate to gauge the GI biogeography and precisely regulate their virulence programs. The EHEC Cra/KdpE/FusR signaling cascade plays a crucial role in this regulation. The relationship between EHEC and different members of the microbiota varies. Our studies using a representative member of each of the main phyla, Bacteroides thetatiotaomicron (Bacteroidetes), Enterococcus faecalis (Firmicutes) and commensal E. coli (Proteobacteria) suggest that EHEC virulence expression varies in response to these commensals, as well as to different combinations of them. In this grant proposal we aim to address how different minimal microbiota compositions impact enteric infections. These studies will build from reductionist to holistic approaches to delve into mechanistic aspects of pathogen-microbiota-host interactions. It is notable that these studies will also be relevant to other enteric pathogens, such as Salmonella enterica and Clostridium difficile, among others, which share several of these pathogen-microbiota interaction strategies with EHEC. Hence the specific aims of this proposal are: Specific Aim 1. Investigate the impact of different members of the microbiota in EHEC?s Cra/KdpE/FusR signaling cascade. Specific Aim 2. Investigate the impact of different microbiota compositions on EHEC infection of enteroids. Specific Aim 3. Investigate the impact of different microbiotas in C. rodentium murine infections.